Whole Gel Eluterand

Mini Whole Gel Eluter

Instruction Manual

Catalog Numbers165-1250165-1251165-1255165-1256

For Technical Service Call Your Local Bio-Rad Off ice or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Table of ContentsPage

Section 1 General Information....................................................................................11.1 Introduction ................................................................................................................11.2 Specifications .............................................................................................................11.3 Safety ..........................................................................................................................2

Section 2 Description and Assembly of Components ...............................................32.1 Whole Gel Eluter Components ..................................................................................42.2 Vacuum Harvester Components ................................................................................5

Section 3 Assembly and Operation.............................................................................63.1 Preparative Slab Gel Electrophoresis.........................................................................63.2 Whole Gel and Mini Whole Gel Eluter Assembly....................................................63.3 Running the Whole Gel Eluter.................................................................................103.4 Harvesting Eluate .....................................................................................................10

Section 4 Optimizing Conditions ..............................................................................124.1 Buffer Selection........................................................................................................124.2 Running Conditions .................................................................................................134.3 Optimization Procedure ...........................................................................................14

Section 5 Maintenance and Sterilization..................................................................145.1 Cleaning the Whole Gel Eluter ................................................................................145.2 Sterilization of the Whole Gel Eluter.......................................................................145.3 Cleaning the Vacuum Harvester ..............................................................................14

Section 6 SDS-PAGE ..................................................................................................156.1 Reagents for SDS-PAGE Slab Gels (Laemmli buffer system)3 .............................156.2 Preparing SDS-PAGE Gels of any %T ...................................................................166.3 Separating Gels - Calculating %T............................................................................176.4 Stacking Gel - 4%T (0.125 M Tris pH 6.8).............................................................17

Section 7 Native PAGE...............................................................................................187.1 Native PAGE Slab Gels ...........................................................................................187.2 Reagents for Discontinuous Native PAGE (Ornstein-Davis) .................................187.3 Prepare Ornstein-Davis Acrylamide Gels ...............................................................197.4 Reagents for Continuous Native PAGE ..................................................................197.5 Prepare Continuous Gels (10 ml acrylamide monomer solution)...........................217.6 Sample Preparation ..................................................................................................21

Section 8 Troubleshooting Guide ..............................................................................21

Section 9 Equipment and Accessories ......................................................................22

Section 10 References ...................................................................................................22

Section 1General Information

1.1 IntroductionThe Whole Gel Eluter provides an alternative method for recovering samples from SDS

and Native PAGE slab gels. It is a preparative electrophoresis instrument which allows simul-taneous electro-elution of multiple protein bands separated on a polyacrylamide gel. Elutionis in the transverse direction, through the thickness of the gel. Proteins are eluted into narrowchambers with each of these fractions containing individual or multiple protein bands.

The eluter consists of a solid acrylic base, a drop-in bottom electrode, an elution cham-ber core, an upper plate/spring electrode and a lid. The Whole Gel Eluter comes in two sizes.The large format accommodates slab gels up to 20 cm long and the small format is designedfor mini-gels (Mini-PROTEAN® II and Ready Gels). Filter paper and a cellophane sheet areplaced between the bottom electrode and the elution chamber core. The gel is sandwichedbetween the top of the elution chamber core and the spring electrode. During elution, the pro-teins are captured in one of the 30 elution chambers of the large eluter or one of the 14 cham-bers of the mini eluter.

One important application for this instrument is the purification and subsequent directscreening of protein mixtures. Andersen and Heron describe a direct cellular analysis of acomplex protein mixture for biological activity.1 The eluter acts as an electrodialyzer remov-ing SDS from protein, leaving them in a non-toxic physiological buffer that can then be useddirectly in a cellular assay. The Rotofor® and Prep cell are recommended for larger scale elec-trophoretic protein purification.

Simple procedures are provided in this manual for optimizing elution conditions. It isrecommended that these procedures be performed for each new sample and when makingchanges to slab gel electrophoresis conditions.

1.2 SpecificationsConstruction

Base acrylic/polycarbonate insertElution chamber core acrylic/polycarbonate insertBottom electrode platinum coated titanium plateUpper plate/spring electrode acrylic/platinum coated titanium plateLid polycarbonateCutting template acrylic

Consumables Large MiniUpper filter paper 50 sheets 50 sheetsLower filter paper 75 sheets 50 sheetsSealing tabs 50 tabs 50 tabsCellophane membrane 25 sheets 25 sheets

Other Specifications Large MiniShipping weight 9.25 lb 4.75 lbOverall size 5.0” H x 6.0” W x 6.0” D 5.0” H x 10.5” W x 10.5” DVoltage limit 300 volts 200 voltsPower limit 15 watts 10 wattsElution sample volume ~3 ml ~0.5 ml

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1.3 SafetyPower to the Whole Gel Eluter is to be supplied by an external DC power supply. This

power supply must be ground isolated in such a way that its DC voltage output floats withrespect to ground. The recommended power supply for this instrument is the PowerPac 300.The maximum specified operating parameters are:

Whole Gel Eluter Mini-Whole Gel Eluter

300 VDC 200 VDC15 W 10 W

The lid assembly provides a safety interlock for the unit. Current flow to the cell is bro-ken when the lid assembly is removed. Do not attempt to circumvent this safety interlock andalways turn off and disconnect the power supply when working with the cell.

The recommended power supplies are ground isolated by design to minimize potentialshock hazard. However, working around high voltage equipment in a laboratory environmentis potentially dangerous. It is the user’s responsibility to always exercise care in setting upand running electrophoresis instruments. Always turn off the power supply before removingthe lid. Do not attempt to use the cell without the safety lid.

Important:

This Bio-Rad product is designed and certified to meet EN61010-1 safety standards.Certified products are safe to use when operated in accordance with the instruction manual.This instrument should not be modified or altered in any way. Alteration of this instrumentwill:

• Void the manufacturer’s warranty

• Void the EN61010-1 certification, and

• Create a potential safety hazard

Bio-Rad is not responsible for any injury or damage caused by use of this instrument forpurposes other than those for which it is intended or by modifications of the instrument notperformed by Bio-Rad or an authorized agent.

This product conforms to the class A standards for Electromagnetic Emissions, intendedfor laboratory equipment applications. It is possible that emissions from this product mayinterfere with some sensitive appliances when placed nearby or on the same circuit as thoseappliances. The user should be aware of this potential and take appropriate measures to avoidinterference.

* EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.

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Section 2Description and Assembly of Components

Whole Gel Eluter exploded view.

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Lid

Upper plate/spring electrode

Upper chamberfilter paper

Acrylamide gel

Sealing tabs

Elutionchamber core

Cellophane

Lower chamberfilter paper**

Lower plateelectrode

Base

** Mini Whole Gel Eluter is assembled with2 sheets of lower chamber filter paper.

2.1 Whole Gel Eluter Components

Base and Lower Electrode

The base forms a stable foundation for the eluter. The recess in it houses the lower elec-trode, the lower filter paper, the cellophane membrane and the elution chamber core.Adjustable feet enable leveling of the eluter.

Elution Chamber Core

The elution chamber core is a multi-channel collection vessel which is dropped into thebase plate and screwed down over the filter paper and cellophane membrane. Proper sealingof the core is important for preventing cross-contamination of the fractions. Proteins elutedfrom the gel are held in the individual chambers until they are collected with the vacuum har-vester or transfer pipet.

Upper Plate/Spring Electrode

Attached to the upper plate is the spring electrode. The electrode makes contact with theupper filter paper. The tension on the spring holds the slab gel in place and seals the tops ofthe elution chambers.

Clamps

The upper plate and base plate are held together by clamps on the sides of the eluter. Toopen the clamps of the Whole Gel Eluter, pull the bottom outward as shown in Figure A.Then disengage the clamp as shown in Figure B. The Mini-Whole Gel Eluter has its clampsmounted in the opposite direction.To open the clamps of the Mini-Whole Gel Eluter, pullthe top outward. Then disengage the clamp at the bottom. To close the clamps, reverse theseprocedures.

A. Step 1

B. Step 2

Opening Whole Gel Eluter Clamps

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Lid

The lid covers the electrical contacts during elution to provide electrical insulation. Thelid cannot be removed without breaking the electrical circuit. It can be placed on the eluter inonly one alignment, so that the anode and cathode connections cannot be accidentally reversed.

Cutting Template

The cutting template is a guide for excising the portion of the slab gel intended to be elut-ed. It also functions as a template for cutting your own upper and lower filter paper and cel-lophane membrane.

Ruler

The ruler has been calibrated to the distance between elution channels. It can be used asa guide to approximate where individually resolved protein bands may elute.

In order to purify an individual protein, the nearest contaminant must be at least 5 mmaway on a preparative gel for the Mini Whole Gel Eluter or 6 mm for the Whole Gel Eluter.

2.2 Vacuum Harvester Components

* The Mini Whole Gel Eluter uses 14, 1.5 ml microcentrifuge tubes.

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Vacuum tubing

Harvest needlearray

30, 12 x 75 mmtubes *

Harvesting lid

Vacuum controlvalve

Harvestingtube rack

Tube Rack

The tube rack forms a stable base for the harvester as well a housing for the collectiontubes. The rack is numbered for easy identification of fractions.

Lid and Needle Array

The lid fits onto the base only one way for proper alignment of the harvester needles withthe collection tubes. Connected to the lid by tubing is the needle array. The needle array insertsinto the harvest ports on the left side of the eluter. Thumb screws hold the needle array inplace, forming a seal between the array and the eluter. The array is designed to be inserted intothe eluter collection ports only one way for proper alignment during harvesting.

Vacuum Control Valve

The vacuum control valve is attached to the lid of the harvesting unit. The valve can begradually adjusted to increase or decrease the vacuum preventing uneven harvesting and splat-tering of eluates.

Section 3Assembly and Operation

3.1 Preparative Slab Gel ElectrophoresisThe Whole Gel Eluters can accommodate SDS or Native PAGE preparative gels up to 3

mm thick. Gel thickness can have a major effect on the elution and recovery efficiency ofproteins. In addition, proteins not eluted into a buffer containing SDS, may adhere to the cel-lophane (or dialysis) membrane. The thicker the gel, the longer it will take to elute the pro-teins and the longer they will be in contact with the membrane. Protein recovery can be greatlyimproved by reversing the current for 10 to 15 seconds after elution and by keeping elutiontimes down to a minimum.

1. Run the SDS or Native gel with a preparative comb following standard electrophoresisprocedures. Refer to Sections 6–7 and References for information on running SDS andNative PAGE gels. The Mini Whole Gel Eluter requires a preparative gel with a minimalwidth of 6.5 cm and the large format eluter requires a minimal width of 14.5 cm. Thelength of the gel is not critical. It is acceptable for some unused elution chambers to be leftempty. It is convenient to have a lane of prestained standard proteins adjacent to thepreparative well to facilitate alignment of the cutting template, but this lane must be cutoff before placing the gel in the eluter.

2. Make up a liter of elution buffer for the Whole Gel Eluter and 500 ml for the Mini WholeGel Eluter. This buffer will be used to equilibrate the preparative slab gel and for the sub-sequent eluter assembly. Refer to Sections 4.1 and 7.4 for elution buffer selection.

3. Following electrophoresis, remove the gel from the gel cassette and equilibrate it in elu-tion buffer for 15–20 minutes. This is to allow an exchange of buffers and minimizeswelling of the gels during elution.

3.2 Whole Gel and Mini Whole Gel Eluter AssemblyOne of the most important factors in the successful use of the Whole Gel Eluter is the

assembly of the unit. As with blotting techniques, it is vital to remove and (or) prevent bub-ble formation under the gel. Second, proper alignment of the protein bands parallel to the elu-tion chambers enhances selectivity.

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1. Place the base of the eluter on a level surface. Place the leveling bubble in the center ofthe base and use the adjustable feet to level the base. Insert the bottom electrode plate inthe depression in the base. Align the electrode with the holes for the screws and electrodestem.

2. Moisten two or three sheets of precut lower chamber filter paper with elution buffer andlay them on top of the bottom electrode plate. The mini eluter requires two sheets andthe large eluter requires three sheets of filter paper to seal the bottoms of the elution cham-bers.

3. Wet a precut sheet of cellophane (or dialysis) membrane and place it on the blotting paper.Work out all air bubbles with the roller provided.

Wet a precut sheet of cellophane and place it on the precut, wetted filter paper.

Using the roller, work out all air bubbles.

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4. Insert the elution chamber core, aligning the holes for the screws and the electrode stem.Finger tighten the hex screws in numerical order (crisscross pattern) while push-ing down on the core. (See diagram below.)

Finger tighten the hex screws in numerical order (criss-cross pattern) while pushing downon the core.

5. Turn the assembly upside down over a sink and shake out any excess buffer squeezedinto the buffer overflow channel during the tightening of the screws.

6. Fill the elution chamber with elution buffer so as to cover all of the channels. Carefullyremove any bubbles trapped in or around the channels.

7. Lay the preparative gel on a clean flat surface and place the cutting template on top of it.Use the template to outline the portion of the gel to be eluted. The template is made of clearacrylic and has numbered lines with which to align the template with the dye front andmolecular markers. Wear clean gloves when handling the gel.

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8. Carefully excise the part of the gel within the template with a razor. (See diagram below.)Use a downward, chopping motion when cutting the gel.Drawing the blade throughthe gel will cause it to tear. It is very important to insure parallel alignment of the proteinbands with the chambers of the eluter for sharply defined and reproducible fractionationof proteins. With experience, it should be possible to achieve a high degree of gel-to-gelreproducibility.

Carefully excise the part of the gel within the template with a razor.

9. Starting at one end, gently lay the excised gel in the elution chamber so that the proteinbands are parallel to the channels. Do not introduce air bubbles under the gel.

10. Soak two sheets of precut upper chamber filter paper in the elution buffer, then lay themon top of the gel, working out any air bubbles with the roller provided.

11. Blot any excess buffer and thoroughly dry the two raised areas containing the aspirationports. Seal these ports with the tabs provided or use transparent tape folded back on itselfto form a tab. (See diagram below.)

Sealing tabs cover all elution ports before running the eluter.

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Sealing tab

12. Thread the tabs through the cutouts of the upper electrode/clamping assembly. (See dia-gram below.) Place the upper electrode assembly on top of the chamber, aligning theholes for the screw heads and the stem of the bottom electrode. Gently push down whileclosing the side clamps.

Confirm that the sealing tabs are threaded through the cutouts of the upperelectrode/clamping assembly.

3.3 Running the Whole Gel Eluter1. Place the lid on top of the assembled eluter so that it is fully enclosed. Note that the lid can

be placed in only one orientation, so that the anode and cathode connections cannot bereversed.

2. Attach the electrical leads to a suitable power supply with the proper polarity.

3. Apply power to the Whole Gel Eluter to begin the elution. As a safety precaution, do notexceed the voltage and power limits. See Section 4.2 for specific running conditions.

4. Turn off the power at the end of the elution time. Reverse the polarity of the electrical leadsat the power supply. Apply reverse current to the Whole Gel Eluter for 10–15 seconds todislodge proteins bound to the cellophane (or dialysis) membrane.

3.4 Harvesting Eluate

Vacuum Harvesting

1. After elution is complete, turn off the power supply, disconnect the electrical leads andremove the lid. Do not disassemble the eluter.

2. Remove the harvest port sealing tabs from both sides of the eluter. Grasp the tabs andgently pull them away from the eluter. Pulling too quickly will disturb the samples. Somespraying from the neighboring holes is normal and not caused by too much elution buffer.

3. For the large format harvester, load the rack with thirty 12 x 75 mm test tubes. Use four-teen 1.5 ml microcentrifuge tubes with separate press on or screw on lids for the mini-for-mat harvester. Tubes with caps attached to them will not fit in the rack.

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Sealing tabs

Upper electrodeclamping assembly

4. Place the lid on top of the harvester. Note that the lid is keyed to the base for proper align-ment of harvester needles and collection tubes.

5. Attach the harvester to a house vacuum or other vacuum source in the off position. Insertthe needle array into the harvest ports on the left hand side of the Whole Gel Eluter(unnumbered side). Tighten the thumb screws on both sides of the needle array so that thegasket seals against the harvest ports. Note that the needle array is keyed for proper align-ment.

6. With the vacuum control valve fully open, turn on the vacuum source. Slowly close thecontrol valve to draw a gentle vacuum with which to aspirate the eluates. Aspirationshould be done slowly to prevent splattering and uneven harvesting.It may be nec-essary to slightly tilt the eluter toward the collection ports to facilitate complete aspirationof the eluates.

** Thirty tubes of eluate from the Whole Gel Eluter are collected in the Harvesting Box and fourteentubes are collected from the Mini-Whole Gel Eluter.

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Vacuum controlvalve

Harvesting box**

Manual Harvesting

1. If a vacuum harvester is not available, eluates may be removed with the proper sizeBio-Rad Disposable Plastic Transfer Pipet (DPTP). The numbered harvest ports on theright side of the eluter are used for manual aspiration of samples. The mini format eluterrequires Bio-Rad DPTP style M (catalog number 223-9563) and the large format eluterrequires Bio-Rad DPTP style D (catalog number 223-9523).

Eluates are collected manually with a Disposable Plastic Transfer Pipet (DPTP).

Section 4Optimizing Conditions

4.1 Buffer SelectionSDS-PAGE or Native PAGE preparative gels can be eluted on the Whole Gel Eluter

using denaturing (SDS) or non-denaturing elution buffer systems. Virtually any buffer canbe used if it proves appropriate for the proteins under investigation, which must be deter-mined empirically. Buffers used for elution are not limited to those described in the fol-lowing sections.

SDS-PAGE

Preparative SDS-PAGE gels can be eluted with either a denaturing or non-denaturingbuffer. The advantage of using a non-denaturing buffer is that the electro-elution processstrips much of the SDS off the proteins, leaving proteins in a non-toxic buffer that can beused directly in a cellular assay. See the Table in Section 7.4 for a list of non-denaturing elu-tion buffers. The addition of SDS to the elution buffers has the advantage of preventing pro-teins from adhering to the cellophane (or dialysis) membrane and insuring completeelectro-elution of all proteins.

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Native PAGE

Preparative Native PAGE gels are usually eluted with non-denaturing buffer systems (seeSection 7.4 for buffers useful in non-denaturing elution). The buffer initially used in runningthe preparative Native PAGE gel should be the first non-denaturing buffer tried for elution.

In contrast to SDS-PAGE where proteins migrate according to size only, the mobilitiesof proteins in native PAGE systems depends on both their charges and sizes. There is no sin-gle elution/electrophoresis buffer system that will optimally elute all native proteins. Whenselecting conditions for the elution of native proteins it is important to consider the pI of theprotein under investigation relative to the pH of the buffer system.

The most important consideration for optimum elution/electrophoresis of a protein is thepH of the buffer. The pH of the buffer must be within the pH range over which the proteinsunder study are stable and retain biological activity. In addition, the pH of the chosen buffersystem must impart sufficient charge to the protein for it to move through the gel at a rea-sonable rate during the run.

The pH of the elution buffer determines the charges (and shapes) of proteins. Therefore,during native elution/electrophoresis, the pH of the buffer will affect the migration of the pro-teins. For example, a buffer with an alkaline pH value relative to the pI of a particular pro-tein will impart net negative charge to the protein. In such a buffer system, the protein migratestoward the positive electrode (anode). Elution buffers with acidic pH values relative to the pIof a protein impart net positive charge to the protein so that it migrates toward the negativeelectrode (cathode). A buffer with a pH value identical to the pI of a protein results in netneutral charge on the protein and it will not move at all in an electric field. Buffers with pHvalues far from the pI of the proteins result in fast elution/electrophoresis rates.

4.2 Running ConditionsThe McLellan buffers described in this manual are a good starting point when selecting

an elution buffer (Section 7.4).2 Eluting with any one of these buffers will give similar volt-age and current readings based on their similar ionic strengths.

The elution rate of any given protein will vary depending on its pI and the pH of the elu-tion buffer. As a general guide the buffer should be at least two pH units away from the pI ofthe protein to impart sufficient charge to the protein. This must be tempered when the stabil-ity and biological activity of the protein must be maintained by a given pH range. Proteinsbound with SDS will have a net negative charge. Under these conditions elution is best in ahigh pH buffer. Determining the proper elution time will require following the optimizationprocedure detailed below (Section 4.3).

Elution Constant ElutionBuffers Current TimeLarge Eluter 200–250 mA 10–30 minSmall Eluter 75–100 mA 10–30 min

Note: Elution parameters are based on use of the McLellan buffers in the pH range of8.1–10.2 to elute E. Coli lysate from a 1 mm thick 12% SDS-PAGE gel. A total of 200µg of protein was loaded per gel. Elution times will vary with buffer pH, protein pI, gelthickness, and gel porosity.

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4.3 Optimization ProcedureThere are multiple factors that can affect the elution of a protein or mixture of proteins

from a slab gel (i.e.protein pI, buffer pH, molecular weight, gel porosity, gel thickness, etc.).Therefore, for every new sample, change in elution buffer or running conditions, and whenmaking changes to the conditions of the initial preparative slab gel electrophoresis, we rec-ommend doing the following optimization procedure.

1. Run a preparative gel with your sample. Make sure that the parameters used for this gelare the same ones used for subsequent elutions (i.e.denatured or non-denatured gels andgel thickness).

2. Follow Sections 3.1–3.2 for the setup of the Whole Gel Eluter, except excise the prepar-ative gel so that the separated protein bands run perpendicular to the elution chambersand not parallel to them.

3. Run the Whole Gel Eluter according to Section 3.3.

4. Eluted samples are harvested over a time course. On the first time point, turn off thepower supply and harvest one elution chamber with a disposable transfer pipet. Re-estab-lish the current to the eluter after sampling.

5. Repeat step 4 for each time point until all of the chambers have been harvested. Thenassay the samples or total protein.

6. Plotting protein concentration versus sampling time will show the optimum elution timefor the buffer system and set running conditions.

Following every elution run, stain the gel and the membrane with Coomassie® Blue. Thiswill indicate the completeness of the elution and whether proteins were lost due to membranebinding.1

Section 5Maintenance and Sterilization

5.1 Cleaning the Whole Gel EluterThe Whole Gel Eluter should be cleaned after every use. The base plate, bottom elec-

trode, elution chamber core and upper plate/electrode should be washed with a laboratorydetergent (for example, Bio-Rad Cleaning Concentrate, catalog number 161-0722), then rinsedthoroughly with distilled water. The lid with electrical leads should be wiped with a dampcloth and allowed to dry thoroughly before use.

5.2 Sterilization of the Whole Gel EluterThe Whole Gel Eluter is not autoclavable. The unit may be sterilized with 70% ethanol.

Do not attempt to sterilize the lid (ethanol will cause crazing of the plastics). The blottingpaper and cellophane (or dialysis) membrane may be autoclaved if desired.

5.3 Cleaning the Vacuum HarvesterThe harvester and tube rack should be washed with a laboratory detergent, then rinsed thor-

oughly with water. The vacuum tubing and needle array should be thoroughly rinsed as well.Turn the harvester lid upside down and fill it with water. This will allow water to flow throughthe tubing and out the array. Dry the tubing by pulling air through the harvester while it isattached to a vacuum source.

Note: Do not attempt to sterilize the Vacuum Harvester. Use autoclaved or pre-sterilizedtransfer pipets if sterile harvesting is required.

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Section 6SDS-PAGE

6.1 Reagents for SDS-PAGE Slab Gels (Laemmli buffer system) 3

Premixed liquid acrylamide (concentrated solution) and premixed electrophoresis buffersare available (see product information section). Sections 6.2–6.4 describe preparation of poly-acrylamide gels using the following stock solutions.

A. 30% Acrylamide Stock Solution

Acrylamide/Bis (30% T, 2.67% C)

146 g acrylamide (29.2 g/100 ml)

4 g N'N'-Bis-methylene-acrylamide (0.8 g/100 ml)

Dissolve in about 350 ml deionized water.

Make to 500 ml with distilled water. Filter and store at 4 °C in the dark.

Or substitute Bio-Rad’s Preweighed Acrylamide/Bis 37.5:1 mixture or 40%Acrylamide/Bis stock solution.

Caution: Acrylamide monomer is a neurotoxin. Avoid breathing acrylamide dust, donot pipet acrylamide solutions by mouth, and wear gloves when handling acrylamidepowder or solutions containing it. For disposal of unused acrylamide, add bis-acrylamide(if none is present), induce polymerization, and discard the solidified gel.

B. Separating (resolving) Gel Buffer Stock

1.5 M Tris-HCl, pH 8.8

Dissolve 54.5 g Tris base in approximately 150 ml deionized water.

Adjust to pH 8.8 with 1 N HCl (Do not back titrate).

Make to 300 ml with deionized water and store at 4 °C.

C. Stacking Gel Buffer Stock

0.5 M Tris-HCl, pH 6.8

Dissolve 6 g Tris base in approximately 60 ml deionized water.

Adjust to pH 6.8 with 1 N HCl (Do not back titrate).

Make to 100 ml with deionized water and store at 4 °C.

D. Sample Buffer (SDS-reducing buffer)

Deionized water 2.8 ml

0.5 M Tris-HCl, pH 6.8 1.0 ml

Glycerol 2.0 ml

10% (w/v) SDS* 1.6 ml

ß-mercaptoethanol 0.4 ml

0.5% (w/v) bromophenol blue 0.2 ml

Total Volume 8.0 ml

Mix equal volumes of sample buffer and sample, and heat at 95 °C for 4 minutes. SDSreducing buffer is 0.06 M Tris-HCl, pH 6.8, 2% SDS, 5% beta-mercaptoethanol, 25% glyc-erol (w/v) and 0.01% bromophenol blue.

*To make a 10% SDS solution: Dissolve 1.0 gram of SDS in deionized water with gentle stirring, then adjust the volume to 10 ml with water.

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E. 10x Electrode (Running) Buffer, pH 8.3 (Makes 1 Liter)

Tris base 30.3 g

Glycine 144.0 g

SDS 10.0 g

Dissolve and adjust to 1,000 ml with deionized water. DO NOT adjust pH with acid orbase. To make 1 liter of 1x electrophoresis buffer (0.025 M Tris, 0.192 M glycine, 0.1% SDS,pH 8.3) dilute 100 ml of 10x stock with 900 ml of deionized water.

6.2 Preparing SDS-PAGE Gels of Any %TThe following table can be used to prepare Tris-HCl acrylamide gels from 4% to 17 % T.

Sections 6.3 and 6.4 provide detailed formulas for calculating specific gel types.Acrylamide/Bis stock solution of 30% (37.5:1) is used. The amounts listed for the componentsin the table below are based on a total volume of 10 ml. When SDS is included in the samplebuffer and the upper electrophoresis running buffer it can be left out of the gels during theirpreparation.

% T Deionized Gel buffer Acrylamide/Bis solution(acrylamide H2O solution* 30% stock (37.5:1)monomer) (ml) (ml) (ml)

4% 6.15 2.50 1.335% 5.80 2.50 1.676% 5.55 2.50 2.007% 5.15 2.50 2.338% 4.80 2.50 2.679% 4.47 2.50 3.0010% 4.17 2.50 3.3311% 3.80 2.50 3.6712% 3.47 2.50 4.0013% 3.15 2.50 4.3314% 2.80 2.50 4.6715% 2.47 2.50 5.0016% 2.15 2.50 5.3317% 1.80 2.50 5.67

* Resolving Gel buffer - 1.5 M Tris-HCl, pH 8.8

* Stacking Gel buffer - 0.5 M Tris-HCl, pH 6.8

Catalysts 10% APS* TEMED*Resolving Gel 50 µl 5 µlStacking Gel 50 µl 10 µl

* Amounts are per 10 ml gel volume. To make 10% APS, dissolve 100 mg in 1 ml of deionized water. TEMED is used neat.

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6.3 Separating Gels - Calculating %T

Monomer Concentration(% T/2.67% C) %T = x(%)

Acrylamides/Bis - 30% T c mlor for 40% T Stock* or c’ mlDeionized water d ml1.5 M Tris-HCl, pH 8.8 2.5 ml10% Ammonium Persulfate(make fresh daily) 50 µlTEMED 5 µl

Total Monomer 10 ml (volume needed fortwo mini gels)

Determine (c ml) and (d ml) for 10 ml of total monomer:

c ml: Calculate the volume of 30% (or 40%) Acrylamide/Bis stock required for 10 mlof the desired total monomer concentration (x) with the following formula: For30% stock:

c ml = (x%)(10 ml)/(30%) = (x)(0.333) ml

c'ml: *Similarly, for 40% stock solution:

c' ml = (x%)(10 ml)/(40%) = (x)(0.25) ml

d ml: Calculate the volume of water required at the desired total monomer concentra-tion with the following formula:

d ml = (10 ml - 2.5 ml - 50 µl - 5 µl) - c ml = 7.445 ml - c ml

Important: One can prepare any desired volume of monomer solution by multiplying the10 ml recipe by the appropriate multiplying factor.

6.4 Stacking Gel - 4% T (0.125 M Tris, pH 6.8)

Monomer Concentration(% T/2.67% C) %T = x(%)

Acrylamides/Bis - 30% T c mlor for 40% T Stock* or c' mlDeionized water d ml0.5 M Tris-HCl, pH 6.8 2.5 ml10% Ammonium Persulfate(make fresh daily)* 50 µlTEMED 5 µl

Total Monomer 10 ml (volume needed fortwo mini gels)

* To make 10% APS, dissolve 100 mg in 1 ml of deionized water.

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Section 7Native-PAGE

7.1 Native PAGE Slab GelsThe discontinuous buffer system of Ornstein-Davis (Tris/chloride/glycine) should be the

first non-denaturing gel system tried.4 An advantage of discontinuous systems for dilute pro-tein solution is the use of stacking gels to concentrate the sample. However, the stacking phe-nomena encountered in discontinuous systems can cause aggregation of some proteins and thiscan severely interfere with resolution.

The pH attained in the resolving gel of the Ornstein-Davis system approaches pH 9.5,which may be outside the range of stability for some proteins. Alternative discontinuousbuffer systems derived for preparative work can be found in an article by Chrambach andJovin.5 The electrophoresis buffers described in this article span the pH range from pH 3 topH 10.

If discontinuous systems cannot be used because of stacking-induced aggregation, a con-tinuous buffer system will be required. In continuous systems the same buffer is used in theupper and lower electrode chambers and in the gel. McLellan describes various continuousbuffer systems from pH 3.8–10.2 which can be tried.2 See Section 7.4 for preparation of thesecontinuous buffers.

7.2 Reagents for Discontinuous Native PAGE (Ornstein-Davis)

A. 30% Acrylamide Stock Solution

Acrylamide/Bis (30% T, 2.67% C)

146 g acrylamide (29.2 g/100 ml)

4 g N'N'-Bis-methylene-acrylamide (0.8 g/100 ml)

Dissolve in about 350 ml deionized water.

Make to 500 ml with distilled water. Filter and store at 4 °C in the dark.

Or substitute Bio-Rad’s Preweighed Acrylamide/Bis 37.5:1 mixture or 40%Acrylamide/Bis Stock solution.

Caution: Acrylamide monomer is a neurotoxin. Avoid breathing acrylamide dust, donot pipet acrylamide solutions by mouth, and wear gloves when handling acrylamidepowder or solutions containing it. For disposal of unused acrylamide, add bis-acrylamide(if none is present), induce polymerization, and discard the solidified gel.

B. Separating (resolving) Gel Buffer Stock

1.5 M Tris-HCl, pH 8.8

Dissolve 54.5 g Tris base in approximately 150 ml deionized water.

Adjust to pH 8.8 with 1 N HCl (Do not back titrate).

Make to 300 ml with deionized water and store at 4 °C.

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C. Stacking Gel Buffer Stock

0.5 M Tris-HCl, pH 6.8

Dissolve 6 g Tris base in approximately 60 ml deionized water.

Adjust to pH 6.8 with 1 N HCl (Do not back titrate).

Make to 100 ml with deionized water and store at 4 °C.

D. Sample Buffer

Deionized water 4.8 ml

0.5 M Tris-HCl, pH 6.8 1.0 ml

Glycerol 2.0 ml

0.5% (w/v)bromophenol blue 0.2 ml

Total Volume 8.0 ml

E. 10x Electrode (Running) Buffer, pH 8.3 (Makes 1 Liter)

Tris base 30.3 g

Glycine 144.0 g

Dissolve in deionized water and adjust the final volume to 1,000 ml. DO NOT adjust pHwith acid or base. To make 1 liter of 1x electrophoresis buffer (0.025 M Tris, 0.192 M glycine,pH 8.3) dilute 100 ml of 10x stock with 900 ml deionized water.

7.3 Prepare Ornstein-Davis Acrylamide GelsUse the following table to prepare gels with %T ranging from 4%T to 10%T.

% T Deionized Gel buffer Acrylamide/Bis solution(acrylamide H2O solution* 30% stock (37.5:1)monomer) (ml) (ml) (ml)

4% 6.15 2.50 1.335% 5.80 2.50 1.676% 5.55 2.50 2.007% 5.15 2.50 2.338% 4.80 2.50 2.679% 4.47 2.50 3.0010% 4.17 2.50 3.33

* Resolving Gel buffer - 1.5 M Tris-HCl, pH 8.8

* Stacking Gel buffer - 0.5 M Tris-HCl, pH 6.8

Catalysts 10% APS* TEMED*Resolving Gel 50 µl 5 µlStacking Gel 50 µl 10 µl

* Amounts are per 10 ml gel volume. To make 10% APS, dissolve 100 mg in 1 ml of deionized water. TEMED is used neat.

7.4 Reagents for Continuous Native-PAGEIn continuous systems, the same buffer is used in the electrode chambers and in the gels.

Since stacking gels are not commonly employed, proteins migrate in bands at least as wide asthe applied sample. Therefore, the sample volume must be kept at a minimum, i.e., the sam-ple should be at as high a concentration as possible. The mobilities of proteins in continuoussystems are dictated primarily by pH rather than by sieving through the polyacrylamide gel.

19

For this reason, 6% polyacrylamide gels are recommended for most applications. For verylarge proteins 4% or 5% gels may be used.

Continuous and Elution Buffer Systems

McLellan describes various continuous buffer systems from pH 3.8 to pH 10.2 all withthe same ionic strength.2 Use the table below to prepare 5x continuous, non-denaturing buffers.Add both the acidic and basic components to 1 liter of water. Do not adjust pH with acid orbase. If the final pH is outside the listed range, discard the buffer and remake it.

Buffer Basic 5X Acidic 5XpH±0.1 component solution component solution

and MW g/L or ml/L and MW g/L or ml/LBeta-Alanine Lactic Acid

3.8 89.09 mw 13.36 g/L 85% soln. 7.45 ml/L

Beta-Alanine Acetic Acid4.4 89.09 mw 35.64 g/L 17.4 M 11.5 ml/L

GABA Acetic Acid4.8 103.1 mw 41.24 g/L 17.4 M 5.75 g/L

Histidine MES6.1 155.2 mw 23.28 g/L 195.2 mw 29.5 g/L

Histidine MOPS6.6 155.2 mw 19.4 g/L 209.3 mw 31.4 g/L

Imidazole HEPES7.4 68.08 mw 14.64 g/L 238.33 mw 41.7 g/L

Tris EPPS8.1 121.14 mw 19.38 g/L 252.2 mw 37.85 g/L

Tris Boric Acid8.7 121.14 mw 30.29 g/L 61.83 7.73 g/L

Tris CAPS9.4 121.14 mw 36.34 g/L 221.3 mw 44.26 g/L

Ammonia CAPS10.2 14.8 M 12.5 ml/L 221.3 mw 22.13 g/L

To make 1 liter of 1X elution buffer, dilute 200 ml of 5X buffer with 800 ml deionizedwater. The final concentrations of buffer components will be:

Buffer pH Basic Component Acidic Component3.8 30 mM Beta-Alanine 20 mM Lactic Acid

4.4 30 mM Beta-Alanine 40 mM Acetic Acid

4.8 80 mM GABA 20 mM Acetic Acid

6.1 30 mM Histidine 30 mM MES

6.6 25 mM Histidine 30 mM MOPS

7.4 43 mM Imidazole 35 mM HEPES

8.1 32 mM Tris 30 mM EPPS

8.7 50 mM Tris 25 mM Boric Acid

9.4 60 mM Tris 40 mM CAPS

10.2 37 mM Ammonia 20 mM CAPS

20

7.5 Prepare Continuous Gels (10 ml acrylamide monomer solution)

% T Deionized Gel buffer Acrylamide/Bis solution(acrylamide H2O solution* 30% stock (37.5:1)monomer) (ml) (ml) (ml)

4% 6.65 2.00 1.335% 6.30 2.00 1.676% 6.05 2.00 2.00

Catalysts 10% APS* TEMED*Resolving Gel 50 µl 5 µlStacking Gel 50 µl 10 µl

* Amounts are per 10 ml gel volume. To make 10% APS, dissolve 100 mg in 1 ml of deionized water. TEMED is used neat.Below pH 6, TEMED becomes less effective as a catalyst. Between pH 4 and pH 6, increasing the concentration of TEMED5-fold to 10-fold will polymerize the gel.

7.6 Sample PreparationSample buffer for continuous native PAGE is a dilution of the electrophoresis buffer.

The concentration of the sample buffer is generally 1/10 that of the running buffer. Glycerolis added to the sample buffer to a final concentration of 20%.

Section 8Troubleshooting Guide

Problem Cause Solution1. Incomplete elution of a. Air bubble formation a. Refer to Section 3.2.

proteins from the gel. between the anodeand cathode.

b. Buffer or running b. Refer to Section 4.conditions are notoptimized.

2. No detectable proteins a. Insufficient protein a. Increase total proteinin collected fractions load. loaded. Use silver stained(complete elution). SDS-PAGE gels to

analyze individualfractions.

b. Protein adhering to the b. Reverse current forcellophane membrane. 10 seconds after the

elution run. Decrease therun time.

3. Cross-contamination a. Elution chamber core a. Tighten the hex screwsof fraction. not tightened down. in a crisscross pattern.

b. Filter paper or cello- b. Refer to Section 3.2phane membrane (assembly).missing or placed inwrong order.

21

Problem Cause Solution4. Poorly defined fraction- a. Protein bands are a. Carefully align the

ation of proteins. not parallel to the cutting template with theelution chambers. dye front and molecular

markers of the gel whencutting.

5. Running conditions a. Buffer concentration a. Make fresh buffer.outside recommended or pH is incorrect.range.

6. Splattering of sample a. Vacuum is too strong. a. Decrease vacuum.in vacuum harvester.

7. Harvester is collecting a. Vacuum is too strong. a. Decrease vacuum.uneven sample volumes.

Section 9Equipment and Accessories

CatalogNumber Product Description

165-1250 Whole Gel Eluter

165-1251 Whole Gel Eluter with large harvester

165-1255 Mini Whole Gel Eluter

165-1256 Mini Whole Gel Eluter with Mini Harvester

165-1260 Large Harvester

165-1261 Mini Harvester

Consumables

165-1270 Whole Gel Eluter Template

165-1271 Mini Whole Gel Eluter Template

165-1275 Cellophane for Whole Gel Eluter , 25

165-1276 Cellophane for Mini Whole Gel Eluter , 25

165-1277 Sealing Tabs for Whole Gel Eluter , 50

165-1278 Sealing tabs for Mini Whole Gel Eluter , 50

165-1279 Roller , 1

165-1280 Lower Chamber filter paper for Whole Gel Eluter , 75

165-1281 Upper Chamber filter paper for Whole Gel Eluter , 50

165-1282 Lower Chamber filter paper for Mini Whole Gel Eluter , 50

165-1283 Upper Chamber filter paper for Mini Whole Gel Eluter , 50

223-9563 DPTP, non-sterile, style M , 500

223-9523 DPTP, non-sterile, style D , 500

223-9645 Culture tubes, 12 x 75 mm , 5 ml capacity, non-sterile, naturalpolypropylene, 2,000

223-9500 Micro Test Tube, 1.5 ml, polypropylene, natural, capless, 500

22

CatalogNumber Product Description

224-0100 Micro Test Tube, 1.5 ml , conical, with separate O-ring scewcaps,non-sterile, 500

165-1284 Ruler, Whole Gel Eluter

Accessories

165-5050 PowerPac 300 , 100/120 V

165-5051 PowerPac 300 , 220/240 V

165-5054 PowerPac 1000 , 100/120 V

165-5055 PowerPac 1000 , 220/240 V

165-5004 Vacuum Station , 120 V, includes vacuum pump, vacuum, regula-tor, instructions

165-5005 Vacuum Station , 240 V

165-5006 Vacuum Regulator

Section 10References

1. Andersen, P. and Heron, I., Journal of Immunological Methods, 161, 29 (1993).

2. McLellan, T., Analytical Biochemistry, 126, 94 (1982).

3. Laemmli, U. K., Nature, 227, 680 (1970).

4. Ornstein, L. and B. J. Davis, Anal. NY Acad. Sci., 121, 321 (1964).

5. Chrambach, A. and T. M. Jovin, Electrophoresis, 4, 190 (1984).

6. Hames, B. D., (1990), in Gel Electrophoresis of Proteins: A Practical Approach, (ed. D. Rickwoodand B. D. Hames), Oxford University Press, New York.

7. Andrews, A. T., (1986), in Electrophoresis: Theory, Techniques and Biochemical and ClinicalApplications, Clarendon Press, Oxford.

8. Allen, R. C., C. A. Saravis and H. R. Maurer, (1984), in Gel Electrophoresis and IsoelectricFocusing of Proteins: Selected Techniques, de Gruyter, New York.

Coomassie is a trademark of ICI.

23

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